The present invention relates to the field of analytical instrumentation for chemical assays and diagnostics, and to the detection of small quantities of analytes in samples. More specifically, the invention relates to an assay device comprising an optical disk having analyte-specific signal elements disposed readably thereon
2.1 Small Scale Clinical Assays
Until recently, most clinical diagnostic assays for the detection of small quantities of analytes in fluids have been conducted as individual tests; that is, as single tests conducted upon single samples to detect individual analytes. More recently, efficiency and economy have been obtained by designing apparatus for multi-sample preparation and automated reagent addition, and by designing apparatus for rapid analysis of large numbers of test samples, either in parallel or in rapid serial procession. Often, such automated reagent preparation devices and automated multiplex analyzers are integrated into a single apparatus.
Large clinical laboratory analyzers of this type can accurately perform hundreds of assays automatically, or semi-automatically, in one hour. However, these analyzers are expensive and only centralized laboratories and large hospitals can afford them. Such centralization necessitates sample transport, and often precludes urgent or emergent analysis of time-critical samples.
Thus, there exists a strong need for simplified clinical assays that will both reduce the cost of such dedicated analyzers and further their distribution. The limit of such effort is the design of clinical tests suitable for use at the patient bedside or in the patient""s home without dedicated detectors. Blood glucose and pregnancy tests are well known examples.
Although useful tests of this sort have been offered for many years, a major breakthrough was the introduction of solid phase immunoassays and other strip tests since approximately 1980. Most notable are Advance(copyright) test (Johnson and Johnson), RAMP(trademark) hCG assay (Monoclonal Antibodies, Inc.), Clear Blue Easy(trademark) (Unipath Ltd.) and ICON (Hybritech).
Clear Blue Easy(trademark) has all reagents in a laminated membrane and uses conjugated colored latex microbeads as the signal reagent. It uses a capillary migration immunoconcentration format. The ICON is a dual monoclonal sandwich immunoconcentration assay. This assay has been rendered quantitative through the use of a small reflectance instrument. Otherwise, all these methods are only qualitative.
Migration distance can be used as a basis for quantitative assays Commercially available are Quantab(trademark) (Environmental Test Systems), AccuLevel(copyright) (Syva), AccuMeter(copyright) (ChemTrak), Clinimeter(trademark) (Crystal Diagnostics) and Q.E.D.(trademark) (Enzymatics). One of the newest is a thermometer-type assay device (Ertinghausen G., U.S. Pat. No. 5,087,556) that is not yet commercially available. These systems can be used to assay general chemistry analytes, such as cholesterol, as well as blood levels of therapeutic drugs.
One disadvantage, however, of each of these formats is that only one, or a very limited number, of assays can conveniently be performed simultaneously.
To fill the gap between massive analyzers and strips, some small instruments have been developed. The most notable is Eclipse ICA(trademark) (Biotope, Inc.). This device is a bench-top, random-access, automated centrifugal immunoassay and chemistry system. Patient samples are pipetted into cassettes that are placed into a rotor. Sixteen tests can be run in approximately 17 minutes. The results are measured by UV/Visual spectrometry or by fluorometry. Four different types of cassette are needed. Each cassette has a relatively complicated structure.
Despite these developments, there still exists a need for a simple device that can easily be used for multiple quantitative assays, and preferably requiring no specialized detector instrumentation.
2.2 Spatially-Addressable Probe Arrays
Recently, spatially addressable arrays of different biomaterials have been fabricated on solid supports. These probe arrays permit the simultaneous analysis of a large number of analytes. Examples are arrays of oligonucleotides or peptides that are fixed to a solid support and that capture complementary analytes. One such system is described by Fodor et al., Nature, Vol. 364, Aug. 5, 1993. Short oligonucleotide probes attached to a solid support bind complementary sequences contained in longer strands of DNA in liquid sample; the sequence of the sample nucleic acids is then calculated by computer based on the hybridization data so collected.
In the assay system described by Fodor et al., the array is inverted on a temperature regulated flow cell against a reservoir containing the tagged target molecules. In order to distinguish the surface bound molecules, the system requires an extremely sensitive detector.
Accordingly, there remains a need for an economical system to fabricate spatially addressable probe arrays in a simplified format that provides both for ready detection and the ability to assay for large numbers of test substances (i.e. analytes) in a fluid test sample in a single step, or a minimum number of steps, or assay for a single test substance or analyte in a large number of fluid test samples.
2.3 Spatially Addressable Laser-Based Detection Systems
Several devices for consumer electronic use permit spatially addressable detection of digital information. In particular, several formats have been developed based on the information recording potential of differential reflectance and transmittance.
In conventional audio or CD-ROM compact disks, digital informationxe2x80x94or digitally encoded analog informationxe2x80x94is encoded on a circular plastic disk by means of indentations in the disk. Typically, such indentations are on the order of one-eighth to one-quarter of the wavelength of the incident beam of a laser that is used to read the information present on the disk. The indentations on the disk cause destructive interference within the reflected beam, which corresponds to a bit having a xe2x80x9czeroxe2x80x9d value. The flat areas of the disk reflect the laser light back to a detector and the detector gives a value of xe2x80x9conexe2x80x9d to the corresponding bit.
In another convention, a change of intensity of a reflected light gets a value of one while a constant intensity corresponds to zero.
Since the indentations have been formed in the disk in a regular pattern from a master copy containing a pre-determined distribution of bits of xe2x80x9czeroxe2x80x9d and bits of xe2x80x9conexe2x80x9d, the resultant signal received by the detector is able to be processed to reproduce the same information that was encoded in the master disk.
The standard compact disk is formed from a 12 cm polycarbonate substrate, a reflective metalized layer, and a protective lacquer coating. The format of current CDS and CD-ROMs is described by the ISO 9660 industry standard, incorporated herein by reference.
The polycarbonate substrate is optical-quality clear polycarbonate. In a standard pressed, or mass-replicated CD, the data layer is part of the polycarbonate substrate, and the data are impressed in the form of a series of pits by a stamper during the injection molding process. During this process, molten polycarbonate is injected into a mold, usually under high pressure, and then cooled so that the polycarbonate takes on the shape of the mirror image of the mold, or xe2x80x9cstamperxe2x80x9d or xe2x80x9cstampxe2x80x9d; pits that represent the binary data on a disc""s substrate are therefore created in and maintained by the polycarbonate substrate as a mirror image of the pits of the stamper created during the mastering process. The stamping master is typically glass.
Pits are impressed in the CD substrate in a continuous spiral. The reflective metal layer applied thereupon, typically aluminum, assumes the shape of the solid polycarbonate substrate, and differentially reflects the laser beam to the reading assembly depending on the presence or absence of xe2x80x9cpits.xe2x80x9d An acrylic lacquer is spincoated in a thin layer on top of the metal reflective layer to protect it from abrasion and corrosion.
Although similar in concept and compatible with CD readers, the information is recorded differently in a recordable compact disk (CD-R). In CD-R, the data layer is separate from the polycarbonate substrate. The polycarbonate substrate instead has impressed upon it a continuous spiral groove as an address for guiding the incident laser. An organic dye is used to form the data layer. Although cyanine was the first material used for these discs, a metal-stabilized cyanine compound is generally used instead of xe2x80x9crawxe2x80x9d cyanine. An alternative material is phthalocyanine. One such metallophthalocyanine compound is described in U.S. Pat. No. 5,580,696.
In CD-R, the organic dye layer is sandwiched between the polycarbonate substrate and the metalized reflective layer, usually 24 carat gold, but alternatively silver, of the media. Information is recorded by a recording laser of appropriate preselected wavelength that selectively melts xe2x80x9cpitsxe2x80x9d into the dye layerxe2x80x94rather than burning holes in the dye, it simply melts it slightly, causing it to become non-translucent so that the reading laser beam is refracted rather than reflected back to the reader""s sensors, as by a physical pit in the standard pressed CD. As in a standard CD, a lacquer coating protects the information-bearing layers.
Other physical formats for recording and storing information are being developed based on the same concept as the compact disk: creation of differential reflectance or transmittance on a substrate to be read by laser.
One such format is termed Digital Video Disc (DVD). A DVD looks like standard CD: it is a 120 mm (4.75 inch) disk that appears as a silvery platter, with a hole in the center for engaging a rotatable drive mechanism. Like a CD, data is recorded on the disc in a spiral trail of tiny pits, and the discs are read using a laser beam. In contrast to a CD, which can store approximately 680 million bytes of digital data under the ISO 9660 standard, the DVD can store from 4.7 billion to 17 billion bytes of digital data. The DVD""s larger capacity is achieved by making the pits smaller and the spiral tighter, that is, by reducing the pitch of the spiral, and by recording the data in as many as four layers, two on each side of the disc. The smaller pit size and tighter pitch require that the reading laser wavelength be smaller. While the smaller wavelength is backward compatible with standard pressed CDS, it is incompatible with current versions of the dye-based CD-R.
The following table compares DVD and CD characteristics:
Thus, a single sided/single layer DVD can contain 4.7 GB of digital information. A single sided/dual layer DVD can contain 8.5 GB of information. A Dual sided/single layer disk can contain 9.4 GB of information, while a dual sided, dual layer DVD contains up to 17 GB of information.
Each of the variations consists of two 0.6 mm substrates that are bonded together. Depending on the capacity, the disc may have one to four information layers. In the 8.5 GB and 17 GB options, a semi-reflector is used in order to access two information layers from one side of the disc.
For the 8.5 GB DVD and 17 GB options, the second information layer per side may be molded into the second substrate or may be added as a photopolymer layer. In either case, a semi-reflector layer is required to allow both information layers to be read from one side of the disk. For the 17 GB DVD, it is necessary to produce two dual-layer substrates, and bond them together.
The DVD laser reader is designed to adjust its focus to either layer depth so that both of them can be quickly and automatically accessed.
All three of the above-described formats require that the platter be spun. The nominal constant linear velocity of a DVD system is 3.5 to 4.0 meters per second (slightly faster for the larger pits in the dual layer versions), which is over 3 times the speed of a standard CD, which is 1.2 mps.
Near-field optical storage disks (TeraStor, San Jose, CA) offer even higher density information storage than DVD. In such devices, the reading head is as close as 150 nm from the disk, and the pit size and track pitch are also of nanometer scale.
Holographic data storage disks offer perhaps the highest known data storage density. Holographic recording exploits three spatial dimensions.
Despite the spatial addressability and high information density of optical media, these media have not previously been thought useful for detection of analytes.
2.4 Waveguide Detection
Waveguides have been used for chemical detection at least since 1982,U.S. Pat. No. 4,608,344, Re. 33,064, incorporated herein by reference. Absorbing and nonabsorbing analytes can be observed with waveguides. The exponential decay of the evanescent wave in uncoated waveguides is sensitive to the absorbance and the refractive index of the surrounding medium. This also affects the intensity of the light that is transmitted by the waveguide. Existing applications of waveguides to detection of analytes show poor spatial resolution.
The present invention solves these and other problems in the art by providing an assay device for detecting analyte, comprising an optical disk having analyte-specific signal elements disposed readably thereon. The optical disk may be read, and the analyte detection thus performed, using optical disk readers useful for reading digitally-encoded information, such as those capable of reading audio CD disks, CD-ROM disks, DVD disks, DIVX disks, laser disks, near-field storage disks, or holographic data storage disks.
In preferred embodiments, the analyte-specific signal elements are disposed readably with the optical disk""s tracking features: that is, the analyte-specific signal elements are readable by the optics used for tracking, although modified or additional optics are not thereby precluded.
In a preferred embodiment of the assay device, the analyte-specific signal elements are cleavable.
In a particularly preferred embodiment, the cleavable signal element comprises: a cleavable spacer having a substrate-attaching end, a signal-responsive end, and a cleavage site intermediate the substrate-attaching end and the signal-responsive end. The cleavable signal element further includes a signal responsive moiety attached to the cleavable spacer at its signal responsive end.
A first side member (also termed side element or side arm) adapted to bind a first site on a chosen analyte, and a second side member adapted to bind a second site of the same analyte, are present on the signal element. The first and second side members confer analyte specificity upon the cleavable signal element.
The first side member is attached to the cleavable spacer intermediate the signal responsive end and cleavage site, and the second side member is attached to the cleavable spacer intermediate the spacer""s cleavage site and substrate attaching end.
Binding of the chosen analyte simultaneously to the first and second side members of a cleavable signal element tethers, or constrains, the signal-responsive moiety to the signal element""s substrate-attaching end, despite subsequent cleavage at the cleavage site that lies intermediate the first and second side members; conversely, failure to bind the chosen analyte simultaneously to the first and second side members of a cleavable signal element permits loss, through cleavage, of that signal element""s signal-responsive moiety. The presence or absence of signal after contact with sample and contact with cleavage agent signals the presence or absence of analyte, respectively.
Typically, the signal responsive moiety of the cleavable signal element is adapted to reflect, scatter, or absorb incident light, particularly incident laser light. In preferred embodiments, the signal responsive moiety is a metal microsphere, and most preferred, a gold microsphere, most preferably a gold microsphere of diameter between 1-3 xcexcm. These embodiments are suitable for detection in existing optical disk readers, such as those used to read audio CD, CD-ROM, DVD, laser disks, near-field optical disks, or the like.
Whether cleavable or no, the analyte-specific signal elements are disposed in or on the assay device in a spatially-addressable pattern.
In another aspect, the invention provides a method of assaying for analyte, comprising the steps of contacting the assay device with a sample, and then detecting, using an optical disk reader, analyte-specific signals therefrom.
In preferred embodiments of this aspect of the invention, the method is performed with assay devices in which the analyte-specific signal elements are cleavable, and the method comprises: contacting the assay device with a sample, cleaving the cleavable signal elements, and then detecting the signal responsive moiety of analyte-constrained cleaved signal elements.
In a related aspect, the invention provides a method of using an optical disk reader to assay for analyte. The method comprises the step of detecting, from an optical disk, analyte-specific signal elements disposed readably with the disk""s tracking features. In preferred embodiments, the method comprises detecting analyte-specific signals from an assay device in which the analyte-specific signal elements are cleavable, and signal is detected from analyte-constrained cleaved signal elements.
The invention further provides a method of making an assay device for detecting analyte, comprising: disposing analyte-specific signal elements on an optical disk readably with said disk""s tracking features.
The signaling element, assay devices and assay methods of the present invention are useful both for the detection of a large number of different analytes in a test sample and the detection of a single analyte in a large number of samples, both quantitatively and qualitatively.
Another aspect of the present invention is to adapt existing assay methods to employ the assay devices of the invention, including the cleavable signal element-based assay devices. Generally, an assay adapted to use the cleavable signal element-based assay device of the present invention comprises the steps of: contacting the assay device with a liquid sample, contacting the assay device with a cleaving agent adapted to cleave said plurality of attached cleavable signal elements, and detecting the presence of the signal responsive moiety of analyte-restrained cleaved signal elements adherent to the solid support substrate.
The spatial addressability of signal elements on the assay device permits identification of analytes bound to distinct signal elements, including identification of multiple analytes in a single assay.
The invention thus provides, in one preferred embodiment of this aspect, nucleic acid hybridization assays, in which the first and second side members of the cleavable signal elements include oligonucleotides. Simultaneous binding of a nucleic acid present in the assay sample to the first and second side members of the cleavable signal element prevents loss, through cleavage, of the signal element""s signal-responsive end.
In another aspect, the invention provides an assay device comprising cleavable signal elements responsive to a plurality of nucleic acid sequences. This aspect of the invention provides a device and method suitable for sequencing nucleic acid through the spatial addressability of signals generated upon contact with a sample containing nucleic acid.
The invention further provides immunoassays. In these embodiments, the specificity-conferring side members of the cleavable signal elements include antibodies, antibody fragments, or antibody derivatives. Simultaneous binding of an analyte to the antibody of the first side member and the antibody of the second side member prevents the loss, through cleavage, of the signal element""s signal-responsive end.
The invention also provides chemical detection assays, in which properly chosen reactive groups on a first and second side member react specifically with functional groups on the chosen analyte to secure the signal responsive moiety to the assay device substrate.
The invention further provides means for detecting electromagnetic radiation. Extremely high resolution X-ray pictures can be exposed and stored on the disk in a format suitable for direct reading on an optical disk reader, such as a CD-ROM or DVD reader, or the like. Other wavelengths of the electromagnetic spectrum are analogously detectable.
The invention also provides means for the detection and counting of cells, and for measuring their dimensions and shapes. In these embodiments, specificity-conferring recognition molecules are disposed upon the assay device substrate. The cells adhere thereto, and are detectable upon binding of signal responsive moieties conjugated to a second cellular recognition molecule Cell recognition molecules include antibodies, receptors, ligands, and adhesion molecules.
In another aspect, the invention provides assay devices that further comprise encoded digital information in the form of computer software.
Another aspect of the present invention provides a monitoring device, comprising an optical disk having a plurality of analyte-specific signal elements, wherein the optical disk is adapted to function as an optical waveguide and the analyte-specific signal elements are so disposed that specific binding of analyte detectably alters the light-transmitting properties of said optical waveguide. This device is suitable for continuous, or repeated, monitoring for presence of analyte. In preferred embodiments of this aspect of the invention, the analyte-specific signal elements are cleavable.
The invention further provides a method of monitoring for presence of analyte, comprising: contacting the monitoring device with a sample, and then detecting alterations in the light-transmitting properties of said monitoring device""s optical waveguide. In a related aspect, the invention provides a method of monitoring for presence of analyte, comprising: contacting the monitoring device having cleavable signal elements with a sample, detecting alterations in the light-transmitting properties of said monitoring device""s optical waveguide, cleaving the signal elements, and then detecting the signal responsive moiety of analyte-restrained cleaved signal elements.
The invention further provides assay devices in which the analyte-specific signal elements are disposed on a solid support substrate fashioned other than in a disk. In preferred embodiments of this aspect of the invention, readable by a laser-based optical reader, the signal elements are disposed readably with the support substrate""s tracking and/or addressing features. Additionally, the assay device substrates may be fashioned as strips, cuvettes, test tubes, well plates, slides, gels, magnetic disks, silicon and other chips.
It is another aspect of the present invention to provide a multiwell sample application plate suitable for applying liquid samples in parallel to the assay devices of the present invention. In one embodiment, the sample application device provides a multiwell plate with a renewable surface film.
The invention further provides instrumentation to ensure correct registration of a sample application device and the assay device. The instrument may optionally comprise magnets to facilitate interaction of the sample with the assay site and/or to remove unbound molecules or particles.